Effect of miR-181d targeting CREB1 on the sensitivity of colorectal cancer cells to 5-fluorouracil

LIU Hong; NONG Fei-fei; DENG Shu-ye

doi:10.13820/j.cnki.gdyx.20243716

Abstract：Objective To identify key genes regulating colorectal cancer(CRC)sensitivity to 5-fluorouracil(5-Fu)using a CRISPR/Cas9 library and to explore the role of microRNA-181d(miR-181d)in mediating this process.Methods A genome-wide CRISPR/Cas9 knockout screening system was established in Ls174t CRC cells.Bioinformatics analysis identified miR-181 d as a key regulator of 5-Fu sensitivity.Loss-of-function(miR-181 d knockout or inhibitor)and gain-of-function(miR-181 d mimic)approaches were applied to evaluate cell viability and apoptosis following 5-Fu treatment.Dual-luciferase reporter assays and Western blotting were used to confirm cAMP re-sponsive element-binding protein 1(CREB1)as a target of miR-181 d.Protein expression levels of CREB1,JUN,and FOS were further examined.Results CRISPR/Cas9 screening showed enrichment of sgRNAs targeting miR-181 d in 5-Fu-treated cells.Suppression of miR-181 d(knockout or inhibitor)significantly promoted CRC cell survival and re-duced apoptosis upon 5-Fu exposure(P＜0.05).Conversely,miR-181 d overexpression inhibited cell survival and en-hanced apoptosis(P＜0.05).Dual-luciferase and Western blot analyses confirmed CREB1 as a direct target of miR-181 d.Overexpression of CREB1 promoted CRC cell survival,suppressed apoptosis,and upregulated JUN and FOS protein levels(P＜0.05).In contrast,miR-181d overexpression downregulated CREB1 and suppressed JUN/FOS expression(P＜0.05).Conclusion miR-181d enhances the sensitivity of CRC cells to 5-Fu by targeting and downregulating CREB1,thereby inhibiting the JUN/FOS signaling pathway,reducing cell survival,and promoting apoptosis.miR-181 d may represent a promising therapeutic strategy to improve chemotherapeutic efficacy in colorectal cancer.